Although it is clear that slute transport across the cell membrane is anN importatn determinant in the rate of cellular metabolism, little is known about the molecular details of transport processes. This project is an investigation of active transport in bacteria and our objective is to understand the mechanism of solute uptake at a molecular level. Out specific aims are to find out the following: (1) What are the properties of carrier proteins that catalyze translocation of hydrophilic substrates across the hydrophobic core of the plasma membrane? (2) What is the role of ion (especially proton) gradients and a transmembrane potential in mediating uphill movement of solutes? Our approach to these questions involves study of respiration-coupled active transport in membrane vesicles isolated from Azotobacter vinelandii, a strictly aerobic bacterium. Membrane preparations consisting principally of vesicles with normal (right-side-out) topology transport glucose in the presence of L-malate. However, preparations that contain large numbers of inverted (inside-out) vesicles transport calcium in the presence of D-lactate. Thse systems are to be further studied by: (1) Development of methods for seperation of membrane besicles of normal topology from those with inverted topology. (2) Measurment of the direction and magnitude of the transmembrane potential and pH gradient in normal and inverted vesicles. (3) Testing the ability of artifically imposed potentials and Ph gradients to drive glucose and calcium transport. (4) Determination of the stoichiometry of proton movements linked to glucose and calcium uptake.